The present invention relates to transgenic animals, compositions and methods relating to the characterization of gene function.
Generally speaking, growth factors are proteins that bind to receptors on the cell surface, with the primary result of activating cellular proliferation and/or differentiation. Many growth factors are quite versatile, stimulating cellular division in numerous different cell types; while others are specific to a particular cell-type. More specifically, growth factors are substances, such as polypeptide hormones, which affect the growth of defined populations of animal cells in vivo or in vitro, but which are not nutrient substances. Proteins involved in the growth and differentiation of tissues may promote or inhibit growth, and promote or inhibit differentiation, and thus the general term xe2x80x9cgrowth factorxe2x80x9d includes cytokines, trophic factors, and their inhibitors. Among growth, or neurotrophic, factors presently known are the transforming growth factors (TGF-alpha, TGF-beta, TGF-gamma). Transforming growth factor-beta appears to elicit a variety of responses in many different cell types.
Widespread neuronal cell death accompanies normal development of the central and peripheral nervous systems. Studies of peripheral target tissues during development have shown that neuronal cell death results from the competition among neurons for limiting amounts of survivor factors (xe2x80x9cneurotrophic factorsxe2x80x9d). The earliest identified of these, nerve growth factor (xe2x80x9cNGFxe2x80x9d), is the most fully characterized and has been shown to be essential for the survival of sympathetic and neural crest-derived sensory neurons during early development of both chick and rat. Barde et al., U.S. Pat. No. 5,229,500, describe nucleic acid sequences encoding brain derived neurotrophic factor (xe2x80x9cBDNFxe2x80x9d), as well as the BDNF protein. BDNF is suggested for treating Parkinson""s Disease and Alzheimer""s Disease. Additional uses are for the identification of homologous regions between BDNF and NGF so as to identify and isolate additional members of the NGF family, and also to generate immunogen by antibodies directed toward BDNF or fragments.
Among TGF-beta members are the bone morphogenetic proteins (BMP). The BMPs have been indicated as useful in wound healing, tissue repair, and to induce cartilage and/or bone growth. BMPs have potent effects during embryogenesis. One member, BMP-4, has been shown to have potent ventralizing effects in Xenopus embryos, leading to the differentiation of blood and mesenchyme and inhibiting the formation of dorsal tissues such as notochord, muscle, and nervous system (see, e.g., Jones et al., Development 115:639-647 (1991)). BMP-4 is expressed ventrally in the Xenopus embryo and its expression is increased by ventralizing treatments such as irradiation with ultraviolet light.
During animal development, cells exchange signals with their neighbors that allow specific groups of cells to produce organized, differentiated structures such as limbs, segments or other body parts. Understanding these processes might allow one to manipulate them to regenerate damaged body parts or to diagnose birth defects. The twisted gastrulation protein (TSG) is one of five known secreted proteins required to pattern the dorsal part of the early Drosophila embryo. Unlike the decapentaplegic (DPP) protein that is required to pattern the entire dorsal half of the embryo, TSG is needed only to specify the fate of the dorsal midline cells. When expressed in a ventral stripe of cells, TSG protein can diffuse to the dorsal-most cells and can rescue the dorsal midline cells in tsg mutant embryos. TSG functions in a permissive rather than instructive role to elaborate cell fates along the dorsal midline after peak levels of DPP activity have xe2x80x98primedxe2x80x99 cells to respond to TSG.
Given the importance of growth factors, a clear need exists for identification and characterization of growth factors which can play a role in preventing, ameliorating or correcting dysfunctions or diseases.
The present invention generally relates to transgenic animals, as well as to compositions and methods relating to the characterization of gene function.
The present invention provides transgenic cells comprising a disruption in a BMP gene. The transgenic cells of the present invention are comprised of any cells capable of undergoing homologous recombination. Preferably, the cells of the present invention are stem cells and more preferably, embryonic stem (ES) cells, and most preferably, murine ES cells. According to one embodiment, the transgenic cells are produced by introducing a targeting construct into a stem cell to produce a homologous recombinant, resulting in a mutation of the BMP gene. In another embodiment, the transgenic cells are derived from the transgenic animals described below. The cells derived from the transgenic animals includes cells that are isolated or present in a tissue or organ, and any cell lines or any progeny thereof.
The present invention also provides a targeting construct and methods of producing the targeting construct that when introduced into stem cells produces a homologous recombinant. In one embodiment, the targeting construct of the present invention comprises first and second polynucleotide sequences that are homologous to the BMP gene. The targeting construct also comprises a polynucleotide sequence that encodes a selectable marker that is preferably positioned between the two different homologous polynucleotide sequences in the construct. The targeting construct may also comprise other regulatory elements that may enhance homologous recombination.
The present invention further provides non-human transgenic animals and methods of producing such non-human transgenic animals comprising a disruption in a BMP gene. The transgenic animals of the present invention include transgenic animals that are heterozygous and homozygous for a mutation in the BMP gene. In one aspect, the transgenic animals of the present invention are defective in the function of the BMP gene. In another aspect, the transgenic animals of the present invention comprise a phenotype associated with having a mutation in a BMP gene. In a preferred embodiment, the non-human transgenic animals of the present invention comprise abnormalities in nociception. In another preferred embodiment, the non-human transgenic animals of the present invention demonstrate a decrease in response to pain. In yet another embodiment, the non-human transgenic animals of the present invention demonstrate an anxiety disorder.
The present invention also provides methods of identifying agents capable of affecting a phenotype of a transgenic animal. For example, a putative agent is administered to the transgenic animal and a response of the transgenic animal to the putative agent is measured and compared to the response of a xe2x80x9cnormalxe2x80x9d or wild type mouse, or alternatively compared to a transgenic animal control (without agent administration). The invention further provides agents identified according to such methods. The present invention also provides methods of identifying agents useful as therapeutic agents for treating conditions associated with a disruption of the BMP gene.
The present invention further provides a method of identifying agents having an effect on BMP expression or function. The method includes administering an effective amount of the agent to a transgenic animal, preferably a mouse. The method includes measuring a response of the transgenic animal, for example, to the agent, and comparing the response of the transgenic animal to a control animal, which may be, for example, a wild-type animal or alternatively, a transgenic animal control. Compounds that may have an effect on BMP expression or function may also be screened against cells in cell-based assays, for example, to identify such compounds.
The invention also provides cell lines comprising nucleic acid sequences of a BMP gene. Such cell lines may be capable of expressing such sequences by virtue of operable linkage to a promoter functional in the cell line. Preferably, expression of the BMP gene sequence is under the control of an inducible promoter. Also provided are methods of identifying agents that interact with the BMP gene, comprising the steps of contacting the BMP gene with an agent and detecting an agent/BMP gene complex. Such complexes can be detected by, for example, measuring expression of an operably linked detectable marker.
The invention further provides methods of treating diseases or conditions associated with a disruption in a BMP gene, and more particularly, to a disruption in the expression or function of the BMP gene. In a preferred embodiment, methods of the present invention involve treating diseases or conditions associated with a disruption in the BMP gene""s expression or function, including administering to a subject in need, a therapeutic agent that effects BMP expression or function. In accordance with this embodiment, the method comprises administration of a therapeutically effective amount of a natural, synthetic, semi-synthetic, or recombinant BMP gene, BMP gene products or fragments thereof as well as natural, synthetic, semi-synthetic or recombinant analogs.
The present invention further provides methods of treating diseases or conditions associated with disrupted targeted gene expression or function, wherein the methods comprise detecting and replacing through gene therapy mutated BMP genes.
Definitions
The term xe2x80x9cgenexe2x80x9d refers to (a) a gene containing at least one of the DNA sequences disclosed herein; (b) any DNA sequence that encodes the amino acid sequence encoded by the DNA sequences disclosed herein and/or; (c) any DNA sequence that hybridizes to the complement of the coding sequences disclosed herein. Preferably, the term includes coding as well as noncoding regions, and preferably includes all sequences necessary for normal gene expression including promoters, enhancers and other regulatory sequences.
The terms xe2x80x9cpolynucleotidexe2x80x9d and xe2x80x9cnucleic acid moleculexe2x80x9d are used interchangeably to refer to polymeric forms of nucleotides of any length. The polynucleotides may contain deoxyribonucleotides, ribonucleotides and/or their analogs. Nucleotides may have any three-dimensional structure, and may perform any function, known or unknown. The term xe2x80x9cpolynucleotidexe2x80x9d includes single-, double-stranded and triple helical molecules. xe2x80x9cOligonucleotidexe2x80x9d refers to polynucleotides of between 5 and about 100 nucleotides of single- or double-stranded DNA. Oligonucleotides are also known as oligomers or oligos and may be isolated from genes, or chemically synthesized by methods known in the art. A xe2x80x9cprimerxe2x80x9d refers to an oligonucleotide, usually single-stranded, that provides a 3xe2x80x2-hydroxyl end for the initiation of enzyme-mediated nucleic acid synthesis. The following are non-limiting embodiments of polynucleotides: a gene or gene fragment, exons, introns, mRNA, tRNA, rRNA, ribozymes, cDNA, recombinant polynucleotides, branched polynucleotides, plasmids, vectors, isolated DNA of any sequence, isolated RNA of any sequence, nucleic acid probes and primers. A nucleic acid molecule may also comprise modified nucleic acid molecules, such as methylated nucleic acid molecules and nucleic acid molecule analogs. Analogs of purines and pyrimidines are known in the art, and include, but are not limited to, aziridinycytosine, 4-acetylcytosine, 5-fluorouracil, 5-bromouracil, 5-carboxymethylaminomethyl-2-thiouracil, 5-carboxymethyl-aminomethyluracil, inosine, N6-isopentenyladenine, 1-methyladenine, 1-methylpseudouracil, 1-methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2-methyladenine, 2-methylguanine, 3-methylcytosine, 5-methylcytosine, pseudouracil, 5-pentylnyluracil and 2,6-diaminopurine. The use of uracil as a substitute for thymine in a deoxyribonucleic acid is also considered an analogous form of pyrimidine.
A xe2x80x9cfragmentxe2x80x9d of a polynucleotide is a polynucleotide comprised of at least 9 contiguous nucleotides, preferably at least 15 contiguous nucleotides and more preferably at least 45 nucleotides, of coding or non-coding sequences.
The term xe2x80x9cgene targetingxe2x80x9d refers to a type of homologous recombination that occurs when a fragment of genomic DNA is introduced into a mammalian cell and that fragment locates and recombines with endogenous homologous sequences.
The term xe2x80x9chomologous recombinationxe2x80x9d refers to the exchange of DNA fragments between two DNA molecules or chromatids at the site of homologous nucleotide sequences.
The term xe2x80x9chomologousxe2x80x9d as used herein denotes a characteristic of a DNA sequence having at least about 70 percent sequence identity as compared to a reference sequence, typically at least about 85 percent sequence identity, preferably at least about 95 percent sequence identity, and more preferably about 98 percent sequence identity, and most preferably about 100 percent sequence identity as compared to a reference sequence. Homology can be determined using a xe2x80x9cBLASTNxe2x80x9d algorithm. It is understood that homologous sequences can accommodate insertions, deletions and substitutions in the nucleotide sequence. Thus, linear sequences of nucleotides can be essentially identical even if some of the nucleotide residues do not precisely correspond or align. The reference sequence may be a subset of a larger sequence, such as a portion of a gene or flanking sequence, or a repetitive portion of a chromosome. The term xe2x80x9ctarget genexe2x80x9d (alternatively referred to as xe2x80x9ctarget gene sequencexe2x80x9d or xe2x80x9ctarget DNA sequencexe2x80x9d or xe2x80x9ctarget sequencexe2x80x9d) refers to any nucleic acid molecule or polynucleotide of any gene to be modified by homologous recombination. The target sequence includes an intact gene, an exon or intron, a regulatory sequence or any region between genes. The target gene comprises a portion of a particular gene or genetic locus in the individual""s genomic DNA. As provided herein, the target gene of the present invention is a BMP gene. A xe2x80x9cBMP genexe2x80x9d refers to a sequence comprising SEQ ID NO: 1 or comprising the sequence identified in GenBank as Accession No.: AF292033; GI NO: 9837569. In one aspect, the coding sequence of the BMP gene comprises SEQ ID NO: 1 or comprises the gene identified in Genebank as Accession No.: AF292033; GI NO: 9837569.
xe2x80x9cDisruptionxe2x80x9d of a BMP gene occurs when a fragment of genomic DNA locates and recombines with an endogenous homologous sequence. These sequence disruptions or modifications may include insertions, missense, frameshift, deletion, or substitutions, or replacements of DNA sequence, or any combination thereof. Insertions include the insertion of entire genes, which may be of animal, plant, fungal, insect, prokaryotic, or viral origin. Disruption, for example, can alter or replace a promoter, enhancer, or splice site of a BMP gene, and can alter the normal gene product by inhibiting its production partially or completely or by enhancing the normal gene product""s activity.
The term, xe2x80x9ctransgenic cellxe2x80x9d, refers to a cell containing within its genome a BMP gene that has been disrupted, modified, altered, or replaced completely or partially by the method of gene targeting.
The term xe2x80x9ctransgenic animalxe2x80x9d refers to an animal that contains within its genome a specific gene that has been disrupted by the method of gene targeting. The transgenic animal includes both the heterozygote animal (i.e., one defective allele and one wild-type allele) and the homozygous animal (i.e., two defective alleles).
As used herein, the terms xe2x80x9cselectable markerxe2x80x9d or xe2x80x9cpositive selection markerxe2x80x9d refers to a gene encoding a product that enables only the cells that carry the gene to survive and/or grow under certain conditions. For example, plant and animal cells that express the introduced neomycin resistance (Neor) gene are resistant to the compound G418. Cells that do not carry the Neor gene marker are killed by G418. Other positive selection markers will be known to those of skill in the art.
A xe2x80x9chost cellxe2x80x9d includes an individual cell or cell culture that can be or has been a recipient for vector(s) or for incorporation of nucleic acid molecules and/or proteins. Host cells include progeny of a single host cell, and the progeny may not necessarily be completely identical (in morphology or in total DNA complement) to the original parent due to natural, accidental, or deliberate mutation. A host cell includes cells transfected with the constructs of the present invention.
The term xe2x80x9cmodulatesxe2x80x9d as used herein refers to the inhibition, reduction, increase or enhancement of a BMP function, expression, activity, or alternatively a phenotype associated with a disruption in a BMP gene.
The term xe2x80x9camelioratesxe2x80x9d refers to a decreasing, reducing or eliminating of a condition, disease, disorder, or phenotype, including an abnormality or symptom associated with a disruption in a BMP gene.
The term xe2x80x9cabnormalityxe2x80x9d refers to any disease, disorder, condition, or phenotype in which a disruption of a BMP gene is implicated, including pathological conditions.